In vitro motility assays using fluorescently labeled proteins have helped define the function of individual molecular motors, particularly those that run on filament tracks in the cytoskeleton which comprise either F- actin or microtubules. While these experiments have defined many motor parameters, they do not image the motor molecule directly, rather they record movements of the fluorescent reporter. This proposal will build on the extensive experience obtained over almost 26 years of light microscopy on single molecular motors by in vitro motility assays by developing a cryoelectron microscopy (cryoEM) technique that will enable the direct visualization of active myosin motors themselves, not just their bound labels, as freeze frames of the ensemble action of a population of asynchronously functioning motors. This technique, dubbed The CryoEM Motility Assay will facilitate 3-D imaging of an ensemble of motors first visualized by fluorescent light microscopy and subsequently rapidly frozen for 3-D visualization using cryoEM. In some cases this may enable particular motor molecules to be imaged in 3-D at ~2 nm resolution. Many muscle diseases, particularly those of heart muscle, have been found to be caused by either defects in the function of myosin motors themselves, or defects in the thin filament proteins. Many diseases of non-muscle tissue are due to defects in cytoplasmic myosin motors. While biochemistry has often described the enzymatic defect, it has not been possible to image the active molecules to observe firsthand the altered structure or interaction. The CryoEM Motility Assay seeks to address this deficiency. The enhanced basic understanding of the structure of active motors may lead to therapeutic advances.